Project Summary Premature termination codon (PTC) diseases afflict millions of people worldwide. Treatment of PTC disorders involves the use of therapeutic agents called nonsense suppressors (NonSups) which stimulate the selective binding to the ribosome of a near cognate tRNA at the PTC position, thereby restoring the production of full length functional proteins. Even small extents of readthrough, leading to limited synthesis of a missing essential protein, can provide dramatic clinical improvement, making this approach highly valuable. However, at present there is only one NonSup, ataluren, approved for clinical use and even this approval is quite limited in scope. The experiments we propose focus on elucidating the detailed mechanisms and sites of action by which NonSups promote PTC readthough and misreading, based on the premise that understanding these mechanisms will facilitate the development of safer, more effective therapeutics for the treatment of PTC diseases. To study NonSup mechanisms we have designed and implemented a system for eukaryotic polypeptide translation composed entirely of separately purified components, termed PURE-LITE. NonSup- induced readthrough (NSIRT) can arise from direct effects of NonSup binding to specific components of the protein synthesis apparatus and from less direct effects that include escape from nonsense-mediated mRNA decay (NMD), and alterations in cellular activity levels of the protein synthesis machinery. The use of PURE- LITE allows partial resolution of the complexity of Total-NSIRT by measuring only effects on readthrough resulting from direct NonSup binding to one or more of the PURE-LITE components. Using PURE-LITE we will determine the efficacy of each NonSup in promoting both read-through translation and misreading, the latter giving rise to toxic side effects for some NonSups, in particular aminoglycosides (AGs) and negamycins. Applying both ensemble and single molecule reaction kinetic approaches with fluorescent and/or radioactive assay components, we will determine how the rates of specific substeps within the readthrough and misreading elongation cycles are modulated by NonSups. The specific molecular targets within the translational apparatus of many NonSups are unknown, despite the success of modern structural biology in providing high resolution structures of many ribosome complexes. This may be due to difficulties in achieving sufficient occupancy of NonSups on the ribosome or because some NonSups may target components of the protein synthesis machinery other than the ribosome. For these NonSups we will use biochemical approaches to identify RNA and protein binding sites within the PURE-LITE system. The results of these site-identification experiments will provide approaches and reagents that will be useful in preparing complexes for subsequent high resolution structural studies. Our Specific Aims are to: 1. Determine the mechanisms of NonSup-induced readthrough; 2. Determine the mechanisms of NonSup-induced misreading; and 3. Identify NonSup target sites.